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Acetoxy drug : Protein transacetylase of buffalo liver - Characterization and mass spectrometry of the acetylated protein product

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Author: Kohli, E. · Gaspari, M. · Raj, H.G. · Parmar, V.S. · Sharma, S.K. · Greef, J. van der · Kumari, R. · Gupta, G. · Seema · Khurana, P. · Tyagi, Y.K. · Watterson, A.C. · Olsen, C.E.
Type:article
Date:2004
Institution: TNO Voeding
Source:Biochimica et Biophysica Acta - Proteins and Proteomics, 1, 1698, 55-66
Identifier: 237711
doi: doi:10.1016/j.bbapap.2003.10.004
Keywords: Chemistry Biology · Analytical research · Acetoxy drug · MALDI-TOF · Protein acetylation · Protein mass spectrometry · Transacetylase · 7,8 diacetoxy 4 methylcoumarin · 7,8 dihydroxy 4 methylcoumarin · acyltransferase · coumarin derivative · liver enzyme · monoacetoxymonohydroxy 4 methylcoumarin · peptide derivative · trypsin · unclassified drug · acetylation · animal cell · article · buffalo · catalysis · enzyme analysis · enzyme purification · liver microsome · mass spectrometry · matrix assisted laser desorption ionization time of flight mass spectrometry · model · molecular weight · nonhuman · peptide mapping · polyacrylamide gel electrophoresis · priority journal · Acetylation · Acetyltransferases · Amino Acid Sequence · Animals · Buffaloes · Coumarins · Glutathione Transferase · Liver · Microsomes, Liver · Molecular Sequence Data · Peptides · Protein Structure, Tertiary · Proteins · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization · Bubalus

Abstract

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4- methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K m (1667 μM) and Vmax (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys51, Lys82, Lys 123, Lsy181, Lys191 and Lys210) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions. © 2003 Elsevier B.V. All rights reserved.